Absorption enhancers such as e.g. BHT, BHA or propyl gallate

ABSTRACT

The invention provides a pharmaceutical composition comprising a mixture of (a) an active macromolecular principle, and (b) an aromatic alcohol absorption enhancer chosen from butylated hydroxy toluene, butylated hydroxy anisole and analogues and derivatives thereof, wherein the aromatic alcohol absorption enhancer is present in an amount by weight greater than or equal to that of the active macromolecular principle, and further comprises a pharmaceutical composition comprising a mixture of (a) an active macromolecular principle, (b) an aromatic alcohol absorption enhancer chosen from propyl gallate, butylated hydroxy toluene, butylated hydroxy anisole and analogues and derivatives thereof, wherein the aromatic alcohol absorption enhancer is present in an amount by weight greater than or equal to that of the active macromolecular principle, and (c) a solubilisation aid capable of increasing the solubility of the aromatic alcohol absorption enhancer in aqueous media.

This application is the US national phase of international applicationPCT/GB2004/001650, filed 15 Apr. 2004, which designated the U.S. andclaims priority of GB 0308732.7, filed 15 Apr. 2003, the entire contentsof each of which are hereby incorporated by reference.

The present invention relates to the use of an aromatic alcohol toenhance the uptake of molecules, including biologically activemacromolecules, into the body, suitably across the intestinal wall fromthe lumen of the gut. In particular the present invention relates tonovel pharmaceutical compositions comprising an active macromolecularprinciple to be absorbed into the body, preferably across the intestinalwall.

Hydrophilic aromatic alcohols, in particular aromatic alcohols in whichthe hydroxy group is not attached directly to the aromatic nucleus, suchas phenoxyethanol, phenyl ethanol and benzyl alcohol, have been employedin pharmaceutical practice for many years as solvents and plasticisers,and have a low toxicity profile when administered via various routes,including the. oral route. Those compounds are all liquids at roomtemperature, and can be readily dissolved in aqueous media.

Hydrophilic aromatic alcohols such as phenoxyethanol and relatedcompounds including phenyl ethanol and benzyl alcohol, have a range ofactions on intestinal cells, one of which is that, when present inrelatively high local concentration, aromatic alcohols transientlyincrease the permeability of a barrier layer of intestinal cells.

It is postulated that this is due to the opening of the tight junctionsbetween these cells creating pores through which even large molecules(macromolecules) can pass by diffusion.

Based on the finding that an increase in the permeability of a barrierlayer of intestinal cells is only seen at relatively high localconcentrations of hydrophilic aromatic alcohol, the applicant's researchhas shown that a solution of hydrophilic aromatic alcoholco-administered orally (as an elixir) with a detectable moleculeproduces no enhancement of uptake. It is postulated that this isbecause, before it reaches the absorption site (in the intestine), thehydrophilic alcohol is rapidly diluted in the gastrointestinal tract toa concentration below which it cannot exert its effect. In addition, themolecules whose uptake one is seeking to elicit will also be diluted outbefore the intestine is reached. It has now been found that anotherclass of aromatic alcohols also displays characteristics of permeationenhancers. These compounds have hydroxyl groups attached directly to thearomatic nucleus and an additional substituent in the position para tothe OH group, and typically display antioxidant properties, which may ormay not be related to their ability to act as permeation enhancers.Examples of this class of compounds are propyl gallate, butylatedhydroxy toluene (BHT) and butylated hydroxy anisole (BHA). Surprisingly,although these materials have been employed routinely in pharmaceuticalpractice for at least twenty years primarily in lipid-basedformulations, generally as antioxidants, the observation that thesematerials are capable of acting as permeation enhancers has never beenmade. This is probably because these compounds are all solids which aresparingly soluble in water, thus making it difficult to incorporate theminto water-based pharmaceutical formulations in high concentrations, andalso preventing them from being available in soluble form to act asenhancers at elevated concentration when the formulation is dispersed inthe lumen of the intestine, or close to any other mucosal surface wherepermeation enhancement is required.

The use of gallate esters or specifically propyl gallate has beendescribed in U.S. Pat. No. 6,180,666 and U.S. Pat. No. 5,962,522respectively as enhancers of bioavailability of small molecules via amechanism in which the propyl gallate inhibits the activity ofcytochrome P450 (in particular CY3PA, located in the endoplasmicreticulum), thereby reducing the metabolic degradation of smallmolecules on their passage through intestinal cells (known as thetranscellular route). Propyl gallate and other esters of gallic acidappear to be potent inhibitors of cytochrome P450, and it is claimedthat sufficient propyl gallate can be introduced into a formulation toexert a significant effect without the need for solubilisation aids.However, the enzyme inhibitor mechanism of action described for propylgallate, however, cannot be expected to enhance the bioavailability ofmacromolecules, since macromolecules are incapable of entering unaidedinto intestinal cells, and so would not come into contact with theendoplasmic reticulum where the enzyme is located. Furthermore,macromolecules such as peptides and proteins are far less susceptible tothe action of cytochrome P450 than are small drug molecules, so thatdegradation by this enzyme is not a major cause of the poorbioavailability of macromolecules from the gut, or other mucosaltissues. A much greater barrier is simply the size of the moleculesthemselves, which prevents them from entering into or passing throughthe cells lining mucosal tissues unaided, where cells which line thesetissues form a continuous impassable wall.

It has now been found that, surprisingly, aromatic alcohols such aspropyl gallate, BHT, BHA and analogues and derivatives thereof arecapable of enhancing the passage of macromolecules across mucosalbarriers by increasing the physical permeability of the mucosal cells.One possible mechanism for this to occur is by transient opening of thetight junctions in between these cells, creating channels along whichthe macromolecules can pass (paracellular route). An alternative mode ofaction is enhancement of fluid-phase pinocytosis, resulting ininternalisation of bulk fluid together with macromolecules withinvacuoles, which are transported from one side of the cell to the other.While yet other mechanisms still not clearly understood are alsopossible, it is considered unlikely that macromolecules actually gaindirect access to the internal cytoplasmic compartment of the cells. Ithas been found that this phenomenon is concentration-dependent, and thatprovision of the aromatic permeation enhancer in the high concentrationsincreases the effect in vivo. Consequently, the use of solubilisationaids is advantageous for these compounds, particularly in the case ofpropyl gallate, to be able to enhance the bio-availability ofmacromolecules from mucosal tissues.

It has now also been discovered that there are certain agents, knownhere as solubilisation aids, which can be used to assist in solubilisingthese aromatic alcohol permeation enhancers, and which, furthermore, canincrease their solubility, and/or rate of dissolution when exposed toaqueous media. This is clearly important if these materials are to exerttheir maximal effect as permeation enhancers.

The invention provides a pharmaceutical composition comprising a mixtureof:

(a) an active macromolecular principle; and

(b) an aromatic alcohol absorption enhancer chosen from butylatedhydroxy toluene, butylated hydroxy anisole and analogues and derivativesthereof, wherein the aromatic alcohol absorption enhancer is present inan amount by weight greater than or equal to that of the activemacromolecular principle.

The invention further provides a pharmaceutical composition comprising amixture of:

(a) an active macromolecular principle; and

(b) an aromatic alcohol absorption enhancer chosen from propyl gallate,butylated hydroxy toluene, butylated hydroxy anisole and analogues andderivatives thereof, and

(c) a solubilisation aid capable of increasing the solubility of thearomatic alcohol absorption anhancer in aqueous media, wherein thearomatic alcohol absorption enhancer is present in an amount by weightgreater than or equal to that of the active macromolecular principle.

The invention also provides the use, in a pharmaceutical composition, ofan aromatic alcohol chosen from butylated hydroxy toluene, butylatedhydroxy anisole and analogues and derivatives thereof as an enhancer forthe absorption of macromolecules into the body.

In a further embodiment the invention provides the use of an aromaticalcohol chosen from butylated hydroxy toluene, butylated hydroxy anisoleand analogues and derivatives thereof in the manufacture of a medicament(pharmaceutical composition) containing an active macromolecularprinciple, in order to enhance absorption of the active macromolecularprinciple into the human or animal body.

The invention also provides the use, in a pharmaceutical composition, ofan aromatic alcohol chosen from propyl gallate, butylated hydroxytoluene, butylated hydroxy anisole and analogues and derivatives thereoftogether with a solubilisation aid capable of increasing the solubilityof the aromatic alcohol absorption enhancer in aqueous media as anenhancer for the absorption of macromolecules into the body.

In a further embodiment, the invention provides the use of an aromaticalcohol chosen from propyl gallate, butylated hydroxy toluene, butylatedhydroxy anisole and analogues and derivatives thereof together with asolubilisation aid capable of increasing the solubility of the aromaticalcohol absorption enhancer in aqueous media in the manufacture of amedicament (pharmaceutical composition) containing an activemacromolecular principle, in order to enhance absorption of the activemacromolecular principle into the human or animal body.

The aromatic alcohol absorption enhancer may be propyl gallate or ananalogue or a derivative thereof, and, preferably is propyl gallate.Suitable analogues and derivatives of propyl gallate include esters ofgallic acid. The esters may be linear or branched chain C₁₋₁₂ alkyl,C₁₋₁₂ alkyloxy, C₁₋₁₂ alkylthio or C₂₋₁₂ alkenyl esters. The compoundsare optionally substituted with halogen, linear or branched chain C₁₋₁₂alkyl, C₁₋₁₂ alkyloxy, C₁₋₁₂ alkylthio or C₂₋₁₂ alkenyl esters. Thearomatic alcohol absorption enhancer may also be chosen from BHT, BHAand analogues and derivatives thereof Suitable analogues and derivativesof BHT or BHA include analogues and derivatives of hydroxy toluene orhydroxy anisole where the methyl group or the methoxy group linked tothe aromatic ring and/or the hydrogen ortho to the hydroxyl group arereplaced by linear or branched chain C₁₋₁₂ alkyl, C₁₋₁₂ alkyloxy, C₁₋₁₂alkylthio or C₂₋₁₂ alkenyl, either unsubstituted or substituted in anyposition, especially by halogen atoms. Preferably, the aromatic alcoholabsorption enhancer is chosen from propyl gallate, BHT and BHA.

The aromatic alcohols disclosed above which are used in pharmaceuticalpractice as antioxidants are included at concentrations up to 0.1% w/vof the total formulation (see entries for individual compounds in theHandbook of Pharmaceutical Excipients, Eds Wade & Weller, ThePharmaceutical Press, London UK, 2^(nd) edition 1994). It is generallyconsidered that higher concentrations of the compounds give no addedantioxidant benefit, and it is thus standard pharmaceutical practice torestrict the concentration of the antioxidants in formulations to nogreater than 0.1%. When used as absorption enhancers according to thepresent invention, however, the efficacy of these compounds isconcentration dependent up to a much higher level, and their proportionsin a pharmaceutical formulation are much higher than previouslydescribed in the prior art.

To the applicant's knowledge, there is no suggestion in the prior art ofthe use of these agents as antioxidants in pharmaceutical formulations.None of these agents play any role in enhancing absorption ofmacromolecules by the oral route, or that these agents may be includedin formulations at levels higher than is standard pharmaceuticalpractice for antioxidants.

For example, EP-A-0295941 discloses a formulation for oraladministration in which BHA, BHT or PG may optionally be included, sothat it is clear that their presence is not essential for biologicalefficacy of the formulation. No concentrations of these agents arespecified, and the formulation is intended as a controlled-releasedosage form, in marked contrast to the present invention where immediatedissolution is desirable to ensure rapid release from the capsule.

WO-A-0222158 provides compositions comprising cyclosporin (not amacromolecule) and containing BHA, BHT and PG generally as antioxidants.Although no specific concentrations of the antioxidants are given, theuse of the compounds as antioxidants suggests a level of no greater than0.1% wt.

U.S. Pat. No. 5,756,450 discloses compositions comprising low molecularwater insoluble compounds including water insoluble polypeptides,especially cyclopeptides such as the cyclosporins. BHA or BHT may beincluded as antioxidants, again in very small quantities.

U.S. Pat. No. 5,342,625 again discloses compositions comprisingcyclosporins. A solubilisation aid may be present to help form amicroemulsion pre-concentrate of the cyclosporin. BHA or BHT may bepresent at low levels as antioxidants.

BHA and BHT may also be present as antioxidants in the compositions ofU.S. Pat. No. 3,996,355 which comprise any drug which is stable in thepresence of a vegetable oil vehicle, more specifically water-sensitivedrugs having a bitter taste. Macromolecules are not envisaged.

Suitable solubilisation aids include, but are not limited to, bile acidsor salts such as sodium taurocholate or taurodeoxycholate, benzylalcohol, phenyl ethanol, phenoxyethanol, transcutol or isopropanol.

The active macromolecular principles falling within the scope of theinvention include all molecules capable of having a beneficial effectwhen absorbed into the human or animal body, especially through theintestinal wall. The beneficial effect may be, for example, therapeutic,cosmetic or preventative such as prophylactic or contraceptive. Theactive macromolecular principles can be of natural (biological),synthetic or semi-synthetic origin.

Macromolecules are preferably defined as molecules having a molecularweight of over 1000 Da, preferably over 2000 Da and most preferably over3000 Da. Examples of macromolecules, including macromolecular activemacromolecular principles, include:

1. Polypeptides and proteins such as insulin; calcitonin; human serumalbumin; growth hormone; growth hormone releasing factors; galanin;parathyroid hormone; blood clotting proteins such as kinogen,prothombin, fibrinogen, Factor VII, Factor VIII of Factor IX;erythropoeitins and EPO mimetics; colony stimulating factors includingGCSF and GMCSF; platelet-derived growth factors; epidermal growthfactors; fibroblast growth factors; transforming growth factors; GLP-1;GAG; cytokines; insulin-like growth factors; bone- andcartilage-inducing factors; neurotrophic factors; interleukins includingIL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12; interferons including interferon gamma, interferon-1a, interferonalphas; TNF alpha; TNF beta; TGF-beta; cholera toxin A and B fragments;E. coli enterotoxin A and B fragments; secretin; enzymes includinghistone deacetylase, superoxide dismutase, catalase, adenosinedeaminase, thymidine kinase, cytosine deaminase, proteases, lipases,carbohydrases, nucleotidases, polymerases, kinases and phosphatases;transport or binding proteins especially those which bind and/ortransport a vitamin, metal ion, amino acid or lipid or lipoprotein suchas cholesterol ester transfer protein, phospholipid transfer protein,HDL binding protein; connective tissue proteins such as a collagen,elastin or fibronectin; a muscle protein such as actin, myosin,dystrophin, or mini-dystrophin; a neuronal, liver, cardiac, or adipocyteprotein; a cytotoxic protein; a cytochrome; a protein which is able tocause replication, growth or differentiation of cells; a signallingmolecule such as an intra-cellular signalling protein or anextracellular signalling protein (eg hormone); trophic factors such asBDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, VEGF, NT3, T3 and HARP;apolipoproteins; antibody molecules; receptors in soluble form such asT-cell receptors and receptors for cytokines, interferons or chemokines;proteins or peptides containing antigenic epitopes and fragments; andderivatives, conjugates and sequence variants of any of the above. Theseand other proteins may be derived from human, plant, animal, bacterialor fungal sources, and extracted either from natural sources, preparedas recombinants by fermentation or chemically synthesised.

2. Polynucleotides such as long-chain linear or circular single-,double- or triple-stranded DNA, single-, double- or triple-stranded RNA,oligonucleotides such as antisense DNA or RNA, and analogues thereofincluding PNA and phosphothioate derivates. In one embodiment it ispreferred that the polynucleotides used in the invention contain a CpGmotif. The coding sequence of the polynucleotide may encode atherapeutic product, in particular the coding sequence may encode anextracellular protein (e.g. a secreted protein); an intracellularprotein (e.g. cytosolic, nuclear or membrane protein); a protein presentin the cell membrane; a blood protein, such as a clotting protein (e.g.kinogen, prothrombin, fibrinogen factor VII, factor VIII or factor IX);an enzyme, such as a catabolic, anabolic gastro-intestinal, metabolic(e.g. glycolysis or Krebs cycle), or a cell signalling enzyme, an enzymewhich breaks down or modifies lipids, fatty acids, glycogen, aminoacids, proteins, nucleotides, polynucleotides (e.g. DNA or RNA) orcarbohydrate (e.g. protease, lipase or carbohydrase), or a proteinmodifying enzyme, such as an enzyme that adds or takes chemical moietiesfrom a protein (e.g. a kinase or phosphatase); a transport or bindingprotein (e.g. which binds and/or transports a vitamin, metal ion, aminoacid or lipid, such as cholesterol ester transfer protein, phospholipidtransfer protein or an HDL binding protein); a connective tissue protein(e.g. a collagen, elastin or fibronectin); a muscle protein (e.g. actin,myosin, dystrophin or mini-dystrophin); a neuronal, liver, cardiac oradipocyte protein; a cytotoxic protein; a cytochrome; a protein which isable to cause the replication, growth or differentiation of cells; aprotein which aids transcription or translation of a gene or regulatestranscription or translation (e.g. a transcription factor or a proteinthat binds a transcription factor or polymerase); a signalling molecule,such as an intracellular or extracellular signalling molecule (e.g. ahormone); an immune system protein such as an antibody, T cell receptor,MHC molecule, cytokine (e.g IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, TNF-, TNF-, TGF-), an interferon (e.g. IFN-, IFN-,IFN-), chemokine (e.g. MIP-1, MIP-1, RANTES), an immune receptor (e.g. areceptor for a cytokine, interferon or chemokine, such as a receptor forany of the above-mentioned cytokines, interferons or chemokines) or acell surface marker (e.g. macrophage, T cell, B cell, NK cell ordendritic cell surfacemarker)(eg. CD 1, 2, 3, 4, 5, 6, 7, 8, 16, 18, 19,28, 40, or 45; or a natural ligand thereof), a trophic factor (e.g.BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, VEGF, NT3, T5, HARP) or anapolipoprotein; a tumour suppressor (e.g. p53, Rb, Rap1A, DCC or k-rev);a suicide protein (thymidine kinase or cytosine deaminase); or a generepressor. The proteins and peptides encoded by the polynucleotidesuseful in the invention may be immunogenic i.e. contain an antigenspecific to the activity of the protein against which antibodies aregenerated by the immune system.

The polynucleotide may have control sequences operably linked to thecoding sequence. The control sequences may typically be those of anyeukaryote or of a virus which infects such eukaryotes. Thepolynucleotide may comprise an origin of replication.

The polynucleotides may be chemically modified. This may enhance theirresistance to nucleases or may enhance their ability to enter cells. Forexample, phosphorothioate oligonucleotides may be used. Otherdeoxynucleotide analogs include methylphosphonates, phosphoramidates,phosphorodithioates, N3′P5′-phosphoramidates and oligoribonucleotidephosphorothioates and their 2′-O-alkyl analogs and2′-O-methylribonucleotide methylphosphonates. Alternatively mixedbackbone oligonucleotides (MBOs) may be used. MBOs contain segments ofphosphothioate oligodeoxynucleotides and appropriately placed segmentsof modified oligodeoxy- or oligoribonucleotides. MBOs have segments ofphosphorothioate linkages and other segments of other modifiedoligonucleotides, such as methylphosphonate, which is non-ionic, andvery resistant to nucleases or 2′-O-alkyloligoribonucleotides.

The polynucleotide suitable for use in the invention is preferably in aform in which it is substantially free of or associated with cells orwith cellular, prokaryotic, eukaryotic, nuclear, chromatin, histone orprotein material. It may be in substantially isolated form, or it may bein substantially purified form, in which case it will generally comprisemore than 90%, e.g. (more than or at least) 95%, 98% or 99% of thepolynucleotide or dry mass in the preparation. Thus the polynucleotidemay be in the form of ‘naked DNA’.

3. Polysaccharides such as heparin, low-molecular weight heparin,polymannose, cyclodextrins and lipopolysaccharide.

4. Any or all of the above either separately or in combination with eachother (for example in the form of a heteroconjugate), or with additionalagents.

In preferred embodiments of the invention the active macromolecularprinciple to be absorbed is selected from calcitonin, insulin, lowmolecular weight heparin, erythropoeitin, human growth hormone andparathyroid hormone, particularly calcitonin, insulin and parathyroidhormone.

Depending on the nature of additional excipients employed, thepharmaceutical composition of the invention may be in liquid, solid,semi-solid or gel form. The pharmaceutical composition of the inventionis suitable for administration via any route giving access to differentmucosal tissues such as buccal and sublingual mucosa, the nasal palate,the lungs, the rectum, the intestinal tract (including the large andsmall intestines) and the vagina. In the case of liquid, semi-solid orgel formulations, these may be either anhydrous or aqueous.

Where the intended site of action of the composition of the invention isthe intestine, it is desirable that the composition is enclosed withinan enteric coating which can withstand the stomach, so that thecomponents of the formulation remain together, undiluted and in closeassociation until they reach the tissues of the small intestine orcolon. Such formulations will suitably be anhydrous. Compositions inliquid form will suitably be administered as enteric-coated capsules,while solid formulations may be administered either withinenteric-coated capsules, or in tablet form, preferably as enteric-coatedtablets.

The enteric coating is chosen appropriately to withstand the naturalcondition of the stomach and to become permeable at the desired locationin the intestine. This is preferably determined by the pH conditionswhich modulate along the length of the intestine. Where the site ofaction is the small intestine, it is preferred that the enteric coatingbecomes permeable and releases its contents at a pH from 3 to 7,preferably from 5.5 to 7, more preferably from 5.5 to 6.5. Where theintended site of action is the colon, it is preferred that the entericcoating becomes permeable and releases its contents at a pH of 6.8 orabove.

Suitable enteric coatings are well known in the art and includecellulose acetate, phthalate, shellac and polymethacrylates such asthose selected from the L and S series of Eudragits in particularEudragits L12.5P, L12.5, L100, L100-55, L30 D-55, S12.5P, S12.5 andS100. Suitable plasticisers or wetting agents, such as triethyl citrateand polysorbate 80 may also be included in the coating mixture.

Selection of an appropriate coating for the capsule, which is preferablyan HPMC or gelatine capsule, can readily be made by the person skilledin the art based on their knowledge and the available literaturesupporting the Eudragit products.

Where the intended site of action is the nasal mucosa, the formulationmay be in the form of an aqueous solution or as a dry powder, which canbe administered as a spray.

Where the intended site of action is the rectum, an appropriate methodof administration is as an anhydrous liquid or solid enclosed within acapsular shell, or incorporated into the matrix of an erodiblesuppository.

For vaginal application, adminstration of the formulation in gel form isalso appropriate.

The aromatic alcohol absorption enhancers are preferablywater-insoluble. The enhancer is suitably present in the composition inan amount of from 1 to 40% by weight, preferably from 5 to 35% byweight, more preferably from 10 to 30% by weight.

In the compositions of the invention, the aromatic alcohol absorptionenhancer is present in an amount (by weight) greater than or equal tothat of the active macromolecular principle. This provides an effectiveconcentration of aromatic alcohol absorption enhancer at the intestinalcell barrier layer (intestinal wall) so as to cause enhanced absorptionin the co-presence of a suitable amount of the active macromolecularprinciple which, when absorbed, will exert its normal beneficial effect.The practitioner of the invention would select the amounts of thearomatic alcohol absorption enhancer and active macromolecular principleon the basis of the amount (for example, blood concentration level) ofthe active macromolecular principle concerned which is necessary fortherapeutic efficacy. The weight ratio of aromatic alcohol absorptionenhancer to active macromolecular principle in the mixture contained inthe capsule is suitably at least 1:1, preferably at least 5:1, forexample from 1:1 to 100:1, preferably from 3:1 to 50:1, most preferablyfrom 5:1 to 20:1.

The ratio of solubilisation aid to aromatic alcohol absorption enhanceris suitably at least 1:1, preferably from 1:1 to 10:1, and mostpreferably from 1.5:1 to 5:1.

The absolute amount of the active macromolecular principle would beselected on the basis of the dosage of the substance required to exertthe normal beneficial effect with respect to the dosage regimen used andthe patient concerned. Determination of these amounts falls within themantle of the practitioner of the invention.

In the composition for oral administration it is preferred that thecontents of the capsule comprises a suitable amount of the activemacromolecular principle to achieve its normal therapeutic effect. Forexample, the composition may contain from 0.05 to 50%, preferably from0.1 to 25%, more preferably from 0.1 to 10% by weight of the activemacromolecular principle based on the weight of the capsule contents(not including the capsule itself).

The composition of the invention may further comprise one or more otherabsorption enhancer compounds, for example, medium chain fatty acids andmedium chain monoglycerides.

The composition of the invention may optionally further comprise anyconventional additive used in the formulation of pharmaceutical productsincluding, for example, anti-oxidants, anti-microbials, suspendingagents, fillers, diluents, absorbents, glidants, binders, anti-cakingagents, lubricants, disintegrants, swelling agents, viscosityregulators, plasticisers and acidity regulators (particularly thoseadjusting the intestinal milieu to between 7 and 7.5). Suitable swellingagents include sodium starch glycolate, pregelatinised starch,microcrystalline cellulose, crosprovidone and magnesium aluminiumsilicate or mixtures thereof Sodium starch glycolate and otherpolyaccharide-based swelling agents may be included in an amount of from5 to 10% by weight. Crosprovidone may be included in an amount of from 5to 30% by weight.

The composition of the invention may optionally further compriseadditional active principles which may enhance the desired action of thecomposition in a synergistic fashion. For example, where the activemacromolecular principle is insulin, the composition may also comprisean insulin sensitiser capable of increasing the body's response to theinsulin absorbed. Examples of sensitisers which could be employed inthis fashion are troglitazone, pioglitazone, rosiglitazone and othermembers of the glitazone class of molecules.

In the composition of the invention where the mixture is contained in acapsule or tablet which comprises the aromatic alcohol absorptionenhancer and active macromolecular principle, the formulation ispreferably substantially anhydrous. In more preferred embodiments of theinvention the entire composition is substantially anhydrous.Substantially anhydrous in the context of this invention means less than5%, preferably less than 1% and more preferably less than 0.5% water byweight of the mixture.

The compositions of the invention can, depending on the activemacromolecular principle used therein, be used in the treatment of avariety of conditions and diseases of the human or animal body bytherapy or, alternately, can be used to introduce macromoleculesessential for the diagnosis of diseases and conditions within the humanor animal body. The compositions of the invention are preferablypharmaceutical or cosmetic compositions.

In the compositions of the invention the mixture contained in thecapsule may be a liquid, semi-solid or gel, which is either in the formof a solution or a microparticulate dispersion. That is to say theactive macromolecular principle(s) for absorption are incorporated intothe formulation either in the form of a solution or as amicroparticulate dispersion. Alternatively, the composition may be inthe form of a solid.

The compositions of the invention are suitably produced by preparing asubstantially anhydrous mixture of the active macromolecular principleand the aromatic alcohol absorption enhancer and then optionally fillinguncoated capsules with the mixture and optionally coating them with anappropriate polymer mixture to achieve the desired permeabilityproperties.

The following Examples serve to illustrate the present invention andshould not be construed as limiting.

EXAMPLES Example 1 Effect in Permeabilising Cell Culture Monolayer

Caco-2 cells (a cell line derived from human colon adenocarcinoma) aregrown as a confluent mono-layer on the surface of a porous membrane(pore size 0.4 μm, surface area 0.33 cm²) separating two aqueouscompartments, the upper compartment filled with 200 μl of culturemedium, and the lower compartment containing 600 μl of the culturemedium. Electrical resistance across the mono-layer is measured using anepithelial voltohmeter connected to electrodes inserted into the mediumon either side of the mono-layer in the upper and lower compartments.This trans-epithelial electrical resistance (TEER) is measuredimmediately before, and fifteen minutes after the addition of aromaticalcohols to the upper compartment (typical results are given in thetable below). Four replicates are employed for each compound, whoseconcentrations are shown in the table below. Fall in TEER is consideredindicative of the increased flow of materials (including bulk fluidphase) across the cell monolayer.

Falls of greater than 50% of the initial value are consideredsignificant. Reducing the concentration tends to reduce the effectobserved.

TABLE TEER (ohm · cm²) Concentration Before 15 minutes after Agent(mg/ml) addition addition Propyl gallate 13 494 −0.6 Propyl gallate 0.2667 629 Butylated hydroxy anisole 0.3 564 183 Butylated hydroxy toluene0.3 633 194

Example 2 Preparation of Formulation Containing Insulin, Propyl Gallateand Sodium Taurocholate

Sodium taurocholate in an amount of 150 mg is mixed with 75 mg of propylgallate in a glass vial and 825 μl of distilled water are added.Dissolution at room temperature is not achieved even on prolongedshaking, but after warming with brief sonication in an ultrasonic bath aclear colourless solution is obtained. Bovine insulin in an amount of8.4 mg is added to the solution with mixing, followed by 10 μl ofglacial acetic acid while vortexing the insulin suspension. A clearsolution is rapidly obtained, with a pH of 3.15. The contents of thevial are frozen rapidly with shaking and lyophilised overnight. Thefollowing day a dry solid is obtained. An amount of 10 mg of the solidis weighed into a 2 ml vial and 50 μl of distilled water added. A clearsolution forms rapidly.

Example 3 Preparation of Formulation Containing Insulin, Propyl Gallateand Sodium Taurodeoxycholate

Identical conditions to those described in example 2 are employed usingtaurodeoxycholate instead of taurocholate. The pH of the final solutionbefore drying is 3.36. A clear solution forms rapidly on addition ofdistilled water to the dried solid as before.

Example 4 Preparation of Formulation Containing Calcitonin, PropylGallate and Sodium Taurocholate

Identical conditions to those described in example 2 are employed,except that 2.3 mg of salmon calcitonin is dissolved in distilled water,and the entire solution added to the mixture of sodium taurocholate andpropyl gallate. A clear solution forms rapidly on addition of distilledwater as before.

Example 5 Preparation of Formulation Containing Calcitonin, PropylGallate and Sodium Taurodeoxycholate

Identical conditions to those described in example 4 are employed,except that taurodeoxycholate is employed instead of taurocholate.

Example 6 Preparation of Formulation Containing Parathyroid Hormone,Propyl Gallate and Sodium Taurocholate

Identical conditions to those described in example 4 are employed,except that 0.5 mg parathyroid hormone is employed instead ofcalcitonin.

Example 7 Preparation of Formulation Containing Parathyroid Hormone,Propyl Gallate and Sodium Taurodeoxycholate

Identical conditions to those described in example 6 are employed,except that taurodeoxycholate is employed instead of taurocholate.

Example 8 Preparation of Formulation Containing Parathyroid Hormone,Propyl Gallate and Sodium Taurodeoxycholate

Identical conditions to those described in example 7 are employed,except that the bile salt/PG mixture is dried without addition ofprotein, and parathyroid hormone is added as a dry powder to the dryresidue after lyophilisation.

Example 9 Preparation of Formulation Containing Human Growth Hormone,Propyl Gallate and Sodium Taurodeoxycholate

Identical conditions to those described in example 8 are employed,except that 20 mg of human growth hormone is employed instead ofparathyroid hormone.

Example 10 Preparation of Formulation Containing Calcitonin, PropylGallate and Propylene Glycol

75 mg of propyl gallate is dissolved by vortexing in 200 μl propyleneglycol. 200 μl of the resultant solution is then transferred to a vialcontaining 1 mg of solid calcitonin. The vial is vortexed briefly todisperse the solid, then shaken for one hour at 37° C., giving a clearsolution.

Example 10 Preparation of Formulation Containing Calcitonin, PropylGallate and Benzyl Alcohol

100 mg of propyl gallate is vortexed in 200 μl of benzyl alcohol, givinga clear solution after several minutes at room temperature. 200 μl ofthe resultant solution is then transferred to a vial containing 1 mg ofsolid calcitonin. The vial is vortexed briefly to disperse the solid.

Example 11 Preparation of Formulation Containing Calcitonin, PropylGallate and Transcutol

100 mg of propyl gallate is vortexed in 200 μl of transcutol, giving aclear solution after one minute at room temperature. 200 μl of theresultant solution is then transferred to a vial containing 1 mg ofsolid calcitonin. The vial is vortexed briefly to disperse the solid forone hour at 37° C., giving a clear solution. 200 μl of the resultantsolution is then transferred to a vial containing 1 mg of solidcalcitonin. The vial is vortexed briefly to disperse the solid, thenshaken for one hour at 37° C., giving a clear solution. 100 μl of thesolution is transferred to a fresh vial to which 100 μl of distilledwater is added. All components remain in solution as a single-phaseclear liquid.

Example 12 Preparation of Formulation Containing Calcitonin, ButylatedHydroxytoluene and Transcutol

100 mg of butylated hydroxy toluene is vortexed in 200 μl of transcutol,giving a clear solution after several minutes at room temperature. 200μl of the resultant solution is then transferred to a vial containing 1mg of solid calcitonin. The vial is vortexed briefly to disperse thesolid, then shaken for one hour at 37° C., giving a clear solution. 100μl of the solution is transferred to a fresh vial to which 100 μl ofdistilled water is added, giving a clear opalescent solution at 37° C.

1. A method of enhancing the absorption of an active macromolecularprinciple which is a polypeptide or protein, polynucleotide orpolysaccharide in a patient, which method comprises orally administeringto said patient a composition comprising an aromatic alcohol absorptionenhancer chosen from propyl gallate, butylated hydroxy toluene (BHT),butylated hydroxy anisole (BHA) and analogues and derivatives thereof,wherein the aromatic alcohol absorption enhancer is present in an amountby weight greater than or equal to that of the active macromolecularprinciple, wherein the composition is coated with an enteric coatingwhich becomes permeable at a pH of from 3 to 7, and wherein when thearomatic alcohol is propyl gallate or an analogue or derivative thereof,the composition further comprises a solubilization aid capable ofincreasing the solubility of the aromatic alcohol absorption enhancer inaqueous media.
 2. A method according to claim 1, wherein the compositionenhances the absorption of the active macromolecular principle acrossthe intestinal wall.
 3. A method of enhancing the absorption of anactive macromolecular principle which is a polypeptide or protein,polynucleotide or polysaccharide in a patient, which method comprisesorally administering to said patient an aromatic alcohol chosen frompropyl gallate, butylated hydroxy toulene, butylated hydroxy anisole andanalogues and derivatives thereof together with a solubilization aidcapable of increasing the solubility of the aromatic alcohol absorptionenhancer in aqueous media.
 4. A method according to claim 2, wherein thecomposition comprises less than 5% by weight of water.
 5. A methodaccording to claim 2, wherein the solubilization aid is selected from aconjugated bile acid or salt, benzylalcohol, phenylethanol,phenoxylethanol, transcutol and isopropanol.
 6. A method according toclaim 2, wherein the composition is comprised in a medicament, whichmedicament is provided in the form of a solution, as a microparticulatedispersion or as a solid.
 7. A method according to claim 2, wherein theactive macromolecular principle is a polypeptide or protein.
 8. A methodaccording to claim 7, wherein the active macromolecular principle isinsulin, calcitonin, growth hormone, parathyroid hormone,erythropoeitin, GLP1 or GCSF, or a derivative or analogue thereof,either synthetic or from natural sources, conforming to structuresderived from either human or animal origin.
 9. A method according toclaim 8, wherein the active macromolecular principle is insulin,calcitonin, parathyroid hormone or a derivative or analogue thereof,either synthetic or from natural sources, conforming to structuresderived from either human or animal origin.
 10. A method according toclaim 9, wherein the active macromolecular principle is insulin or aderivative or an analogue thereof, either synthetic or from naturalsources, conforming to structures derived from either human or animalorigin and an insulin sensitizing agent is also present.
 11. A methodaccording to claim 2, wherein the active macromolecular principle is apolynucleotide which is single, double or triple-stranded RNA or apolysaccharide which is heparin.
 12. A method according to claim 11,wherein the polynucleotide is double-stranded RNA.
 13. A methodaccording to claim 1, wherein the active macromolecular principle has amolecular weight of over 2000 Da.
 14. A method according to claim 1,wherein the active macromolecular principle has a molecular weight ofover 3000 Da.